Laser scanner apparatus for fluorescence measurements

ABSTRACT

The invention relates to a laser scanner apparatus ( 1 ) for imaging and/or measuring fluorescent samples which are located on specimen slides ( 8 ) and treated with two different fluorescent dyes and also to a corresponding method. This laser scanner apparatus ( 1 ) comprises a motor-drivable sample table ( 2 ) with a receptacle ( 34 ) for specimen slides ( 8 ) in a sample plane ( 49 ); at least one laser ( 51,52 ) and a first optical system ( 53 ) for providing two laser beams ( 54,55 ) of differing wavelength oriented parallel to each other and extending parallel to this plane ( 49 ); a scanner means ( 72 ) comprising a scanner head ( 50 ) which can move back and forth parallel to this plane ( 49 ) and in a direction of movement ( 75 ) and has an optical deflection element ( 56 ) for deflecting the laser beams ( 54,55 ) toward the sample; and a first objective ( 57 ) for focusing the laser beams ( 54,55 ) on the sample in the plane ( 49 ). The laser scanner apparatus ( 1 ) additionally comprises a second optical system ( 58 ) for forwarding to detectors ( 61,61′ ) emission beam bundles ( 59,60 ) which are triggered by the laser beams ( 54,55 ) on the sample and deflected by the first objective ( 57 ) and the deflection element ( 56 ) in a direction substantially parallel to the plane ( 49 ) and two detectors ( 61,61′ ) for detecting the emission beam bundles ( 59,60 ) of differing wavelength coming from the samples. The optical deflection element ( 56 ) of the laser scanner apparatus ( 1 ) according to the invention comprises a wedge-shaped dichroic mirror ( 62 ) with front and rear dichroic surfaces ( 63,64 ) which are arranged at an intermediate angle (β) to each other. The wedge-shaped dichroic mirror ( 62 ) is adjusted in such a way that the two laser beams ( 54,55 ) are each reflected at one of the surfaces ( 63,64 ). Thus, the wedge-shaped dichroic mirror ( 62 ) causes through the intermediate angle (β) a spatial separation of the two resulting focal points ( 65 ) and the two emission beam bundles ( 59,60 ) guided in the direction of the detectors ( 61,61 ′).

RELATED PATENT APPLICATIONS

This patent application claims priority of the Swiss patent applicationNo. CH 01641/07 filed on Oct. 22, 2007, the entire disclosure of whichis incorporated herein by explicit reference for any purpose.

FIELD OF TECHNOLOGY

The invention relates to a laser scanner apparatus for imaging and/ormeasuring fluorescent samples which are located on specimen slides andtreated with two different fluorescent dyes and also to a correspondingmethod. In this case, this laser scanner apparatus comprises amotor-drivable sample table with a receptacle for specimen slides in asample plane; at least one laser and a first optical system forproviding two laser beams of differing wavelength oriented parallel toeach other and extending parallel to this plane; a scanner meanscomprising a scanner head which can move back and forth parallel to thisplane and in a direction of movement and which comprises an opticaldeflection element for deflecting the laser beams toward the sample andan objective for focusing the laser beams on the sample in the plane.The laser scanner apparatus additionally comprises a second opticalsystem for forwarding emission beam bundles which are triggered by thelaser beams on the sample and deflected by the objective and thedeflection element in a direction substantially parallel to the plane aswell as two detectors for detecting the emission beam bundles ofdiffering wavelength coming from the samples.

RELATED PRIOR ART

Conventional optical scanning microscopes have long been used forimaging fluorescent samples located on specimen slides. Confocal opticalscanning microscopes are being used more and more often owing to theimproved resolution. A microscope of this type is known for example fromGB 2 184 321 A. This microscope guides the light of a laser source alongan optical path in order to scan a sample located in the object plane ofthe microscope with the focused light beam. The fluorescent beam emittedby the sample is guided back through the same optical path for thepurposes of descreening or descanning, separated from the excitationbeam by means of a dichroic mirror and imaged on a confocal openingbefore a detector. Thus, an image is formed from the fluorescence of asample, without the light directed toward the sample for the purposes oftriggering the fluorescence being able to strike the detector.

Many of the commercially available microscopes are based on this designand have beam splitters or filters for breaking down the light emittedby the sample into beams having a differing wavelength range. This alsoallows two fluorescent dyes to be used and the emission thereof to bemeasured using two different detectors.

Nevertheless, all confocal scanner systems which guide the twoexcitation light beams having the two different wavelengths onto thesame scanning spot have the drawback that the two emission signals canbe delimited only spectrally. As the absorption and/or the fluorescenceemission spectra of the dyes which are used usually overlap, it is notpossible (especially in the case of relatively large differences inintensity) to distinguish between them reliably and quantitatively. Sothat first an image having a first fluorescence spectrum and then asecond image having a different type of excitation beam do not have tobe generated in a time-consuming manner, there have been proposedscanning microscopes and “scanner apparatuses” providing at least twodifferently oriented excitation beams.

U.S. Pat. No. 5,304,810 for example discloses a microscope of this typewhich, with two or more illumination beams which are spatially separatedfrom one another, generates two or more illumination points which arespatially separated from one another and scans a sample simultaneouslywith these illumination points. The fluorescence emission beam bundleswhich are spatially separated from one another and simultaneouslygenerated as a result are measured, in accordance with their respectivescan position, simultaneously by means of individual detectors orientedtoward these illumination points which are spatially separated from oneanother. U.S. Pat. No. 6,628,385 B1 also discloses a microscope of thistype which generates by means of two excitation lasers two separatelight spots on a sample. In this case, the two excitation beamspenetrate at slightly different angles an opening in a 45° mirror andthen strike an objective element. This causes the provision of two lightspots which are separate from each other on the sample, an emission beambundles being generated at each light spot. The two resulting emissionbeam bundles are reflected on the 45° mirror and strike a secondarylens, after which they each reach one of two detectors immediately orafter a second deflection. In addition, optical separating elements,such as dichroic filters or prisms, can be positioned before thedetectors which are configured as photomultipliers. A scanning or screensystem arranged between the 45° mirror and the objective element can beused for scanning the samples.

WO 02/059677 A1 discloses an optical system for the excitation andmeasuring of fluorescence on or in samples treated with fluorescentdyes. This system comprises at least one laser for exciting thefluorescent dyes used, a mirror for deflecting the laser light in thedirection of a sample, a deflection element for deflecting the lightfrom the laser onto this mirror in a Y direction of an (in this caseCartesian) coordinate system, optics for imaging a first focal point ofthe laser light on the sample, a scanning unit which comprises themirror and the optics and can move in the Y direction, a sample tablewhich can move in the X and Z directions of the coordinate system fororienting the sample relative to the first focal point, an opticalarrangement for imaging the light emitted by the sample in a holediaphragm arranged in a second focal point and a detector for measuringthe intensity of the light passing through the hole diaphragm.

In addition, these known microscopes for the highly sensitive scanningof samples arranged in a regular pattern (known as an array) are capableof scanning an entire standard specimen slide for light-opticalmicroscopy and operate satisfactorily at medium resolution. It shouldhowever be borne in mind that if the resolution increases, additionaleffects, such as dynamic displacements between the color channels, canbecome visible. As a result, the imaging points of the red and the greenchannel, for example, no longer lay precisely one above the other. Therelative displacement can change dynamically between the channels overthe extension of the image. In addition, this displacement dependssubstantially on the positional precision of the sample in the focus.For these reasons, relative displacement can be correctedretrospectively using software only with great difficulty.

If the two channels are to be separated from each other not onlyspectrally but rather also spatially, the two focal points of theexcitation lasers have to be separated from each other on the sample.The only way to achieve this is for the focused laser beams of the twolasers to strike the scan objective at an angle to one another that issmall but nevertheless significant. It is generally known that all beamsstriking the objective at a specific angle are focused onto the samepoint within the focal plane. A specific angle of incidence before theobjective therefore corresponds in all cases to a specific locationafter the objective. In this connection, it is immaterial whether thelaser beam strikes the objective at the centre thereof or in any otherpartial region of the objective aperture; the focusing at one and thesame focal point is not affected thereby. The beam angle after theobjective is however different; the beams then converge at the focalpoint from different directions. In the exact focal point this makes nodifference, but in planes lying slightly therebelow or thereabove itdoes. There, the beams deviate from the exact focal point at differingspeeds as a function of this angle.

If then two laser beams are to be focused, in accordance with theserequirements, spatially separately from each other on a sample and inthe focal plane and if these laser beams therefore form an angle to eachother as they strike the scan objective, this inevitably means that atleast one of the two laser beams can now also no longer extend, beforestriking the mirror element, precisely parallel to the scan axis.

If the scanner head is then moved, the point at which the laser beamstrikes the objective changes. The beam is still deflected onto the samefocal point, but at different angles. Outside the focal plane, there arethen obtained, in accordance with that which was stated hereinbefore,different positions in accordance with the position of the scanner headin the X direction and in accordance with the deviation of the sampleplane from the exact focal plane in the Z direction. The latterdeviation can never be completely ruled out within realistic apparatustolerances and also cannot be controlled as accurately as desired as arandom tolerance.

Although the described effects are per se small, they are clearly andsignificantly discernible in the exemplary construction at resolutionsbelow 5 μm. The described effects can lead to the images of the twodetection channels not being congruent over the entire image range andto the extent of the deviation varying in an uncontrolled manner overthe image. Quantitative measurements of very small structures are as aresult impossible or at least falsified. Additionally, the errors arediscernible as locally varying color fringes.

The document DE 197 07 227 A1 discloses a light scanning device forexciting and detecting of emission light. The scanning device comprisesa light generating unit, a deflecting unit, an imaging unit, and aconfirmation unit for detection. In order to minimize scanning time at aconstantly kept high resolution, the light scanning device furtherpossesses a separation unit for separating an initial light beam bundleinto at least two resulting light beam bundles. The separation unitcomprises a wedge-shaped dichroic double mirror. The initial light beambundle is reflected at the two surfaces of the wedge and thus separated.Alternatively, two wedge-shaped double mirrors form four resulting lightbeam bundles out of a single initial light beam bundle. In this way,simultaneous scanning of a sample is possible and thus the scanning timereduced.

The document EP 0 490 510 A discloses a sensor arrangement with atelescope in order to align the light of a field of vision with respectto detectors. A wedge-shaped beam splitter with two non-parallelsurfaces reflects the light in the direction of a detection arrangementwith two detectors. In this case, one surface of the beam splitter isembodied as a dichroic layer whereas the other surface is accomplishedas a mirror. The dichroic layer reflects a light bundle of a first wavelength range and allows a light bundle of a second wave length range topass through. The light of the second wave length range is thenreflected at the mirror surface. In this way, the two light bundles oftwo different wave length ranges but of the same field of vision aredirected to two different regions in a single focal plane of aphotodetector arrangement. Starting from a single field of vision, theformation of two different images is thus based on the separation of twodifferent wave length ranges.

OBJECT AND SUMMARY OF THE PRESENT INVENTION

The object of the present invention is to propose an alternative laserscanner apparatus for imaging and/or measuring fluorescent samples whichare located on specimen slides and treated with two differentfluorescent dyes.

According to a first aspect, this object is achieved by a laser scannerapparatus according to the features as disclosed herein below byproposing a laser scanner apparatus for imaging and/or measuringfluorescent samples which are located on specimen slides and treatedwith two different fluorescent dyes, which apparatus comprises:

-   (a) a motor-drivable sample table with a receptacle for specimen    slides in a sample plane;-   (b) at least one laser and a first optical system for providing two    laser beams of differing wavelength oriented parallel to each other    and extending parallel to this plane;-   (c) a scanner means comprising a scanner head which can move back    and forth parallel to this plane and in a direction of movement and    has an optical deflection element for deflecting the laser beams    toward the sample;-   (d) an objective for focusing the laser beams on the sample in the    plane;-   (e) a second optical system for forwarding to detectors emission    beam bundles which are triggered by the laser beams on the sample    and deflected by the objective and the deflection element in a    direction substantially parallel to the plane, and-   (f) two detectors for detecting the emission beam bundles of    differing wavelength coming from the samples.

The laser scanner apparatus according to the invention is characterizedin that the optical deflection element comprises a wedge-shaped dichroicmirror with front and rear dichroic surfaces arranged at an intermediateangle β to each other, the wedge-shaped dichroic mirror being adjustedin such a way that the two laser beams are each reflected at one of thesurfaces, and the wedge-shaped dichroic mirror causing through theintermediate angle β a spatial separation of the two resulting focalpoints and the two emission beam bundles guided in the direction of thedetectors.

According to a second aspect, this object is achieved by a method foroperating a laser scanner apparatus according to the features disclosedherein below by proposing a method for imaging and/or measuringfluorescent samples which are located on specimen slides and treatedwith two different fluorescent dyes, which method includes the followingsteps:

-   (a) providing a motor-drivable sample table with a receptacle for    specimen slides in a sample plane;-   (b) providing two laser beams of differing wavelength oriented    parallel to each other and extending parallel to this plane with at    least one laser and a first optical system;-   (c) deflecting the laser beams toward the sample with an optical    deflection element of a scanner means comprising a scanner head    which is implemented to be movable back and forth parallel to this    plane and in a direction of movement;-   (d) focusing the laser beams on the sample in the plane with an    objective;-   (e) forwarding to detectors emission beam bundles which are    triggered by the laser beams on the sample and deflected by the    objective and the deflection element in a direction substantially    parallel to the plane with a second optical system, and-   (f) detecting the emission beam bundles of differing wavelength    coming from the samples using two detectors.

The method according to the invention is characterized in that theoptical deflection element used is a wedge-shaped dichroic mirror withfront and rear dichroic surfaces arranged at an intermediate angle β toeach other, the wedge-shaped dichroic mirror being adjusted in such away that the two laser beams are each reflected at one of the surfaces,and the wedge-shaped dichroic mirror causing through the intermediateangle β a spatial separation of the two resulting focal points and thetwo emission beam bundles guided in the direction of the detectors.

Additional preferred features according to the invention will emergefrom respectively dependent claims.

Advantages of the laser scanner apparatus according to the inventioninclude:

-   -   Red/green images recorded using the laser scanner apparatus        according to the invention are, despite the high resolution,        almost congruent or can be made congruent for the entire image        by a simple X/Y correction (linear shifting).    -   The wedge-shaped dichroic mirror used in accordance with the        invention, with front and rear dichroic surfaces arranged at an        intermediate angle to each other, allows at the same time        spatial separation of the spots for exciting the fluorescence in        the samples as well as separation of the two emission beam        bundles guided in the direction of the detectors.    -   The pentamirror arrangement used in accordance with the        invention corrects all tilting of the scanner head about an axis        extending at right angles to the scan axis, so that the        resulting focal points do not change their current position in        the sample plane.    -   The counter oscillator used in accordance with the invention        compensates for movement pulses of the rapidly moving scanner        head, so that these pulses are not transmitted to the laser        scanner apparatus.    -   The linear encoder (measuring rod) which is used in accordance        with the invention and placed both in the optical main plane and        in the scanning or screen plane defined by the two scanning        laser beams allows very precise detection of the instantaneous        position of the scanner head and exact calculating back to the        position of the current focal points.

BRIEF INTRODUCTION OF THE ATTACHED DRAWINGS

The laser scanner apparatus according to the invention will be describedhereinafter based on schematic drawings which do not limit the scope ofthe present invention and which illustrate examples of particularlypreferred embodiments. In the drawings:

FIG. 1 is a vertical partial section through two specimen slidemagazines and an object table placed therebefore during the transfer ofa specimen slide from the sample magazine to the object table;

FIG. 2 is a horizontal partial section through the specimen slidemagazines and a plan view onto the object table placed therebeforeduring the transfer of a test specimen slide from the test objectmagazine to the object table;

FIG. 3 shows vertical views of the specimen slide magazine with the testobject magazine opened, wherein

-   -   FIG. 3A is a front view, viewed from the object table, on the        insertion side of the two specimen slide magazines, and    -   FIG. 3B is a vertical section, looking toward the object table,        of the two specimen slide magazines;

FIG. 4 shows vertical partial sections through the object table and thetransverse tilting device thereof, wherein

-   -   FIG. 4A shows the object table looking toward the specimen slide        magazines and with a specimen slide held in a two-way manner in        the closed object table, and    -   FIG. 4B shows the object table looking away from the specimen        slide magazines, with the object table opened, after the removal        or prior to the insertion of a specimen slide;

FIG. 5 is a vertical partial section through the object table and theheight adjustment and longitudinal tilting device thereof;

FIG. 6 is a schematic diagram with basic optical elements of the laserscanner apparatus with a scanner head according to a first embodiment;

FIG. 7 are schematic diagrams of the scanner head, wherein

-   -   FIG. 7A shows a second embodiment of the scanner head, and    -   FIG. 7B shows a third embodiment of the scanner head;

FIG. 8 is a horizontal partial section through a laser scanner apparatuswith basic optical elements, a scanner means with a scanner head and anobject table with specimen slide magazines;

FIG. 9 is a horizontal partial section through the scanner head of thelaser scanner apparatus with the associated displacement transducer;

FIG. 10 is a schematic diagram of the displacement transducer for thescanner head and the non-linear movement thereof during scanning as aX/t diagram which refers to the different periods of time (Δt₁; Δt₂) fordetecting the fluorescence light emanating from an object in accordancewith the position of a number of pixels (Δx) on the X axis;

FIG. 11 shows a test specimen slide having the format of a standardspecimen slide for light-optical microscopy and comprising exclusivelylight-stable test structures; and

FIG. 12 is a vertical section through an eccentric device for adjustingthe focal line.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 is a vertical partial section through two specimen slidemagazines and an object table placed therebefore during the transfer ofa specimen slide from the sample magazine to the object table. These twospecimen slide magazines form part of a laser scanner apparatus 1according to the invention for imaging and/or measuring fluorescentsamples which are located on specimen slides and treated withfluorescent dyes. This laser scanner apparatus comprises a sample table2 defining a sample plane 49 and a motorized transportation device 3 formoving a specimen slide from a storage unit 4 to the sample table 2 andback. In this case, the storage unit 4 comprises a respective samplepart 7, which has at least one respective bearing point 6 and isaccessible during operation of the laser scanner apparatus 1 to thetransportation device 3, for sample specimen slides 8 and test part 9for test specimen slides 10. In this laser scanner apparatus accordingto the invention, the test part 9 is configured separately from thesample part 7 as a test part magazine 9′, which is rigidly connected tothe laser scanner apparatus 1, for one or more test specimen slides 10.As a result, a test specimen slide 10 stored in the test part 9 is notmanually accessible to an operator when the laser scanner apparatus 1 isin operation. This has the advantage that a suitable test specimen slidecan be provided at all times, without the risk of a test specimen slide10 of this type becoming soiled or even damaged as a result of beingmishandled by operators. The test part magazine 9′ illustrated in thepresent case comprises an open insertion side 15.

In the embodiment illustrated in the present case, the sample part 7 isarranged axially above the test part 9, and the test part 9 of thestorage unit 4 is rigidly connected to an adjustment plate 11, which ismoveable relative to the sample table 2 of the laser scanner apparatus1, of the storage unit 4. In this case, the adjustment plate 11 of thestorage unit 4 is displaceable substantially perpendicularly relative tothe sample plane 49 of the sample table 2. Thus, any desired specimenslide 8,10 can be brought to the level of the sample plane 49 defined bythe sample table 2 and provided for linear transportation to the sampletable 2.

Departing from this view in FIG. 1, the adjustment plate can also befixed and thus provide an immovable connection between the laser scannerapparatus 1 and the test part magazine 9′. In such a case, the sampletable 2 has to be moved relative to the test part magazine 9′ if anydesired specimen slide 8,10 is to be transported linearly from a samplepart magazine 7′ or a test part magazine 9′ to the sample table 2. It isalso possible to dispense with an adjustment plate 11 altogether and tofasten the test part magazine 9′ at any location to the laser scannerapparatus 1 in such a way as to render a test specimen slide 10 storedin the test part 9 manually inaccessible to an operator when the laserscanner apparatus 1 is in operation.

Further alternatives (not shown) include moving a test specimen slides10, which is already located in the sample plane 49 of the sample table2, in this sample plane 49 in relation to a stationary sample table 2,moving the sample table 2 in this sample plane 49 in relation to astationary test part magazine 9′ or else mutual movement of the sampletable 2 and test part magazine 9′. All these cases allow lineartransportation of any desired specimen slide 8,10 from a sample partmagazine 7′ or a test part magazine 9′ to the sample table 2. Inaddition, it is conceivable to use a robot which removes a specimenslide 8,10 from one of the magazines 7′,9′ and positions it on thesample table 2; in this case, the magazines 7′,9′ and the sample table 2can assume almost any desired position relative to one another.

It is however preferred for the sample plane 49 of the sample table 2 tobe arranged substantially horizontally, the sample table 2 carrying aspecimen slide 8,10 above it. Nevertheless, the sample table 2 can alsobe arranged overhead, so that the specimen slide 8,10 used is arrangedunder the sample table. Any other desired spatial position of the sampleplane 49 is in principle also conceivable, although less preferred.

The laser scanner apparatus 1 according to the first embodimentillustrated in FIG. 1 comprises preferably a housing 5, the sample part7 being configured as a magazine 7′, which can be inserted from theoutside into the housing 5 of the laser scanner apparatus 1, for a largenumber of sample specimen slides 8. The sample part 7 can be mountedpreferably reversibly on the adjustment plate 11 of the storage unit 4.In the embodiment shown, a plug-in dovetail connection connects thesample part magazine 7′ to the adjustment plate 11 which in this case isvertically movable. Thus, the sample part magazine 7′ can be gripped(manually or by a robot) by the handle 42 and lowered into the housing 5in a substantially vertical direction and plugged into the dovetail 43of the adjustment plate 11. As in this case the two magazines 7′,9′ arearranged perpendicularly one above the other, the test part magazine 9′,which is screwed securely to the adjustment plate 11, is preferablyprecisely the lower stop for the sample part magazine 7′ inserted intothe dovetail 43.

The bearing points 6 in the sample part magazine 7′ and/or in the testpart magazine 9′ are configured for receiving specimen slides havingsubstantially the dimensions of a standard specimen slide forlight-optical microscopy. Preferably, these bearing points 6 areseparated from one another by bearing webs 12, so that these specimenslides are each based on two bearing webs 12 each extendingsubstantially over the entire length of the specimen slides 8,10.

The sample table 2, a vertical section of which is shown in FIG. 1, isconfigured so as to be movable, for transferring sample specimen slides8 or test specimen slides 10, by means of a spindle drive 84 which isarranged on a suspension 83 immediately before a storage unit 4 forspecimen slides 8,10 of this type. The receptacle 34 of the sample table2 comprises preferably two mutually opposing grooves 35 for receivingthe two longitudinal edges 14 of a sample specimen slide 8 or a testspecimen slide 10. The sample plane 49 is in this case preferablyarranged substantially horizontally. The sample table 2 comprises, forsecuring a specimen slide 8,10 in a clamping manner in a directionsubstantially perpendicular to the surface of the specimen slides, twostationary webs 36 and a jaw 37 which can be moved resiliently towardthese webs 36 and has two upright side walls 38 defining, together withthe lower edges of the webs 36, the opening width of the grooves 35 (cf.also FIG. 4).

Preferably, a controller 40 monitors or governs a motor 87 which drivesthe spindle drive 84. As a result, the controller 40 controls themovements of the sample table 2.

FIG. 2 is a horizontal partial section through the specimen slidemagazines shown in FIG. 1 and a plan view onto the object table placedbefore said specimen slide magazines during the transfer of a testspecimen slide from the test object magazine to the object table. Thetest part magazine 9′ illustrated in this case comprises an openinsertion side 15, which can be covered in its breadth at least partlyby a respective flap 16 which extends substantially over the entirestack height of the magazine 9′ and can be swiveled away individually.This flap 16 is in this case swiveled away, thus allowing theillustrated test specimen slide to be slid out from the insertion side15 of the test part magazine 9′ without being impeded by the swivel-awayflap 16 in the process.

In the case of the test part magazine 9′, this swivel-away flap 16 formsa part of the interlocking engagement which prevents a test specimenslide 10 stored in the test part 9 from being manually accessible to anoperator when the laser scanner apparatus 1 is in operation. In the caseof the sample part magazine 7′, this swivel-away flap 16 allows, whenfolded in, handling (for example swiveling or tilting) of a magazine 7′filled with at least one sample specimen slide 8, without the risk ofthese specimen slides falling out. Preferably, both the sample partmagazine 7′ and the test part magazine 9′ have, but at least the samplepart magazine 7′ has on its side opposing the insertion side 15, alocking plate 20 which extends substantially over the entire stackheight and covers part of the breadth of this side. This locking plate20 prevents, especially during handling of the sample part magazine 7′,specimen slides from falling out on the other side.

Preferably, the flaps 16, which can be swiveled away individually ineach magazine 7′,9′, are rotatably fastened to a respective axis 17arranged laterally of the magazines 7′,9′. The flaps 16, which can beswiveled away individually, each comprise an angular plate 18 extendingpreferably substantially over the entire stack height of the magazines7′,9′. These swivel-away flaps 16 are pressed, for releasing theinsertion side 15 of one of the magazines 7′,9′, against the respectivemagazine 7′,9′ preferably by a rotatable, motor-driven eccentric roller19. Variants (not illustrated) include moving the swivel-away flaps 16using a lever, a ram or a slide.

So that the specimen slides 8,10 are positioned in the magazines 7′,9′substantially without play, each of these bearing points 6 comprisespreferably a contact spring 13 which acts resiliently on a longitudinaledge 14 of an inserted specimen slide. In addition, the spring pressureholds the respectively opposing longitudinal edge 14 of the specimenslide 8,10 in a position which is defined by the corresponding magazine7′,9′ and is suitable for defining a reference for the origin of acoordinate system. Likewise, the sample table 2 is equipped preferablywith movable contact parts 39 in the form of rolls (cf. FIG. 2) whichalso secure this longitudinal edge 14 in a defined position, thus againproviding a reference for the origin of the coordinate system.

At least the sample part magazine 7′ comprises, preferably at a corneropposing the insertion side 15, a control opening 21 extendingsubstantially over the entire stack height for establishing the presenceor absence of a specimen slide in a specific bearing point 6. Thepresence or absence of a specimen slide 8,10 in a specific bearing point6 can be established using different methods and devices. Thus, forexample (cf. FIG. 2), a light beam 23 extending substantiallyhorizontally or a light barrier of a control device 22 can be directedobliquely through the magazines 7′,9′, if the control opening 21 ispermeable to this light beam 23. The deflection, scattering or weakeningof the light beam 23 by a specimen slide 8,10 which is present in astorage area 6 can easily be established using a light-sensitive sensor.While FIG. 2 shows a control opening 21 in the form of a “cut-offcorner”, the light beam 23 can also be emitted into the magazines 7′,9′through the insertion side 15 and strike a sensor on the opposite,non-cut-off side; an oblique orientation relative to the direction oftransportation of the specimen slides 8,10 and/or the attachment of adeflection mirror (both not shown) likewise allow detection of thespecimen slides in their magazines even when the sample table 2 has beendrawn close.

Further variants for establishing the presence or absence of a specimenslide in a specific bearing point 6 of one of the two magazines 7′,9′are based for example on the basis of capacitive approximate evidence.

Preferably, the transportation device 3 of the laser scanner apparatus 1comprises a discharging slide 31 which is configured so as to actsubstantially parallel to the sample plane 49 through the side opposingthe insertion side 15 of the magazines 7′,9′ and for transporting asample specimen slide 8 or a test specimen slide 10 out from its bearingpoint 6 and from the insertion side 15 to the sample table 2. Thistransportation device 3 preferably also comprises a charging slide 32which is configured for transporting a sample specimen slide 8 or a testspecimen slide 10 out of the sample table 2 and through the insertionside 15 into a storage area 6 in one of the magazines 7′,9′.Particularly preferably, the charging slide 32 comprises a pivotableflap 33 which can be swiveled upward and thus be moved away via thespecimen slide 8,10 which is inserted in the sample table 2, withoutthis flap 33, which can be tilted about an axis 47, touching thespecimen slide. Thus, this flap can be moved via the specimen slide 8,10and lowered after said specimen slide, whereupon the specimen slide canbe grasped by the flap 33 and drawn out of the sample table 2. Theupward swiveling of the flap 33 allows the sample table 2 and thespecimen slide 8,10 inserted therein to move to the location of thescanner means 72. This upward swiveling of the flap 33 about the tiltaxis 47 thus allows free movement of the sample table 2, without theflap 33 being able to enter into contact with the inserted specimenslide 8,10.

Preferably, the drive 44 for the movable adjustment plate 11, the drive45 for the discharging slide 31 and the drive 46 for the charging slide32 are each an electric motor which is controlled and monitored by thecontroller 40.

The sample table 2 shown in FIG. 2 comprises, for securing a specimenslide 8,10 in a clamping manner, contact parts 39 which can move in adirection substantially parallel to the surface of the specimen slidestoward at least one of the longitudinal edges 14 of the specimen slideand resiliently delimit the opening breadth of the receptacle 34. Inthis case, the contact parts 39, which can move toward at least one ofthe longitudinal edges 14 of the specimen slide, are configuredpreferably as rolls each having a substantially vertical axis.

Preferably, a controller 40 monitors or governs a motor 87 which drivesthe spindle drive 84. As a result, the controller 40 controls themovements of the sample table 2.

FIG. 3 shows vertical views of the specimen slide magazines with thetest object magazine opened. FIG. 3A is in this case a front view,viewed from the object table, of the insertion side of the two specimenslide magazines. The vertically movable adjustment plate 11 can be seenon the right-hand side and its movability is marked by a double-headedarrow. The sample part 7 is arranged just above the test part 9, thesample part magazine 7′, with in this case eight sample specimen slides8 resting in the bearing points 6, being fastened axially above the testpart magazine 9′ to in this case two test specimen slides 10. Theswivel-away flap 16 of the sample part magazine 7′ is closed, while theswivel-away flap 16 of the test part magazine 9′ is opened and releasessubstantially the entire breadth of the insertion side of the test partmagazine 9′. The swiveling-away of the swivel-away flap 16 of the testpart magazine 9′ is brought about in this case by the eccentric roller19 which presses onto the angular plate 18 of this flap. The eccentricroller 19 is arranged preferably at least close to the sample plane 49defined by the sample table 2, so that, despite the verticaldisplacement of the storage unit 4, the correct flap 16 is swiveled awayat all times. It may clearly be seen how the contact springs of the testpart magazine 9′ press resiliently onto one side edge 14 of the testspecimen slides 10.

FIG. 3B is a vertical section, looking toward the object table, of thetwo same specimen slide magazines. The vertically movable adjustmentplate 11 can be seen on the left-hand side and its movability is markedby a double-headed arrow. The sample part magazine 7′ is slid via thedovetail 43 of the adjustment plate 11 and is held in this case by thetest part magazine 9′ in a constant position on the adjustment plate 11.The test part magazine 9′ is in this case screwed tight to theadjustment plate 11. The contact springs 13 of the sample part magazine7′ and the test part magazine 9′ can be seen clearly in this case on theright-hand side of the specimen slide stack.

FIG. 4 shows vertical partial sections through the object table 2 andthe trans-verse tilting device thereof or the tilting mechanism 79 whichcomprises a motor-driven eccentric 80 and a one-sided hinge pin 81. Thistilting mechanism 79 serves to orient a sample or a specimen slide 8,10relative to a focal line 101 extending in a scanning plane 76 (cf. FIG.5). The focus of the first objective 57 and the direction of movement 75of the scanner head 50 of the laser scanner apparatus 1 define thisfocal line 101. This focal line 101 itself defines, together with theoptical deflection element 56 of the scanner head 50, the scanning plane76.

This scanning plane 76 is thus defined by the direction of movement 75of the scanner head 50 and the optical deflection element 56 thereof.This scanning plane 76 stands in this case substantially perpendicularto the sample plane 49. This focal line 101 is defined by the directionof movement 75 of the scanner head 50 and the focal point 65 of thescanner objective 57 and lies, when the apparatus is correctly adjusted,in the sample plane 49. The hinge pin 81 can be configured as an actualaxle (not shown). Preferred, however, is a virtual hinge pin 81 formedby a steel spring 104. This steel spring 104 is screwed onto the sampletable 2 or onto the bearing part 103 preferably by means of a respectiveyoke 105. This steel spring 104 causes a force opposing the eccentric80, thus providing a simple, play-free tilting mechanism for the bearingpart 103 of the sample table 2.

FIG. 4A shows the object table 2 of the laser scanner apparatus 1looking toward the specimen slide magazines 7′,9′ and with a specimenslide 8 held doubled (i.e. in two ways) in the closed object table 2.The sample table 2 comprises a tilting mechanism 79 with a motor-driveneccentric 80 and a one-sided hinge pin 81, which tilting mechanism 79can be used to orient a specimen slide 8,10 or a sample relative to afocal line 101. FIG. 12 is a section through a preferred embodiment ofan eccentric device of this type. This focal line 101 lies preferably inthe sample plane 49 and in a scanning plane 76 which the scanner head 50defines with its optical deflection element 56 and its direction ofmovement 75. In this case, the scanning plane 76 stands preferablyperpendicular to the sample plane 49 (cf. also FIG. 5). The eccentric80, which is preferably motor-driven, can be used to correct thetransverse inclination of the specimen slide 8,10 or the sample table 2,so that the focal line 101 of the scanner means 72 comes to lie exactlyin the sample plane 49.

Preferably, the sample plane 49 is arranged substantially horizontally.The receptacle 34 of the sample table 2 comprises two mutually opposinggrooves 35 (cf. FIG. 4B) for receiving the two longitudinal edges 14 ofthe sample specimen slide 8 shown or a test specimen slide 10 (notshown).

The sample table 2 comprises, for securing a specimen slide 8,10 in aclamping manner in a direction substantially perpendicular to thesurface of the specimen slides, preferably a bearing part 103 with twostationary webs 36. In addition, the sample table 2 comprises a jaw 37which can move resiliently toward these webs 36 and has two upright sidewalls 38. These side walls 38 define, together with the lower edges ofthe webs 36, the opening width of the grooves 35. The movable jaw 37 issupported by springs 30 resiliently relative to the bearing part 103 ofthe sample table 2, so that these springs 30 press the two upright sidewalls 38 of the movable jaw 37 resiliently toward the underside of thespecimen slide 8. As a result, a sample specimen slide or a testspecimen slide 10, which preferably has at least approximately the massof a glass specimen slide for light-optical microscopy, is held in thesample table 2 in a clamping manner in the vertical direction.

The sample table 2 comprises, for securing a specimen slide 8,10 in aclamping manner in a direction substantially parallel to the surface ofthe specimen slides, contact parts 39 which can move toward at least oneof the longitudinal edges 14 of the specimen slide 8 and resilientlydelimit the opening breadth of the receptacle 34. These contact parts39, which can move toward at least one of the longitudinal edges 14 ofthe specimen slide 8, are configured preferably as rolls each having asubstantially vertical axis. The groove 35 opposing the rolls 39 definesa stop of the sample specimen slides 8 or test specimen slides 10 whichis suitable for defining the axis of a coordinate system of the laserscanner apparatus 1.

Also shown dipping into a recess 98 is in this case a dimpling punch 88which, as the sample table 2 and the storage unit 4 draw close,penetrates the sample table and, with this penetration, draws the jaw 37and the side walls 38 away from the webs 36 of the bearing part 103.

FIG. 4B shows the object table 2, looking away from the specimen slidemagazines 7′,9′, with the object table 2 opened, after the removal orprior to the insertion of a specimen slide 8,10. Because at this momentno specimen slide 8,10 is located in the sample table 2, the roll-likecontact parts 39 are in their extreme position. The roll-like contactparts 39 are displaced from this extreme position counter to thepressure of spring elements as soon as a specimen slide 8,10 is insertedinto the sample table 2. It may also clearly be seen in this case howthe dimpling punch 88 strikes a ramp 89, so that the movable jaw 37 ofthe sample table 2 is drawn downward somewhat, thus facilitating theinsertion of a specimen slides 8,10 into the receptacle 34 of the sampletable 2.

FIG. 5 is a vertical partial section through the object table and alsothe height adjustment and longitudinal tilting device thereof. Thesample plane 49 defined by the sample table 2 can be adjusted insubstantially the Z direction (in this case in the vertical direction)in that the linearly displaceable sample table 2, which is linearlyfastened to a suspension 83, rests, together with this suspension 83, ona motor-driven eccentric 106 and is pivotably fastened on one side to aframe 82. FIG. 12 is a section through a preferred embodiment of aneccentric device of this type. If the eccentric 106 is rotated somewhat,the suspension 83 rises or falls accordingly with the sample table 2.This movement allows the plane of the sample table 2, i.e. the sampleplane 49, to be brought into correspondence with the plane of a bearingpoint 6 in the sample part magazine 7′ or in the test part magazine 9′of the storage unit 4, thus allowing a linear transfer to take placebetween one of these magazines 7′,9′ and the sample table. Preferably,the corresponding magazine is provided in the Z direction bydisplacement of the movable adjustment plate 11, so that merely apossible fine adaptation to the eccentric 106 of the sample tablesuspension 83 must take place. The eccentric 106, which is preferablymotor-driven, can be used to correct the longitudinal inclination of thespecimen slide 8,10 or the sample table 2, so that the focal line 101 ofthe scanner means 72 comes to lay exactly in the sample plane 49. Infact, the correction of the longitudinal inclination is also accompaniedby vertical displacement, i.e. along a Z axis.

For the purposes of a specimen slide transfer of this type, the sampletable 2 is preferably drawn as close as possible to the storage unit 4in the substantially horizontal Y direction. As the sample table 2 drawsclose to the storage unit 4, a dimpling punch 88 penetrates the sampletable 2 and accordingly lowers a support of the receptacle 34 of thesample table 2 for receiving a specimen slide. As a result, the sampletable 2 is provided for receiving a specimen slide 8,10. Thisdrawing-close takes place preferably by means of a spindle drive 84which is mounted on the suspension 83 and along a linear guide 85. Thespindle drive 84 is connected to the motor 87 via a flexible coupling86, so that exact linear guidance of the sample table 2 in substantiallythe Y direction can take place even when the sample plane 49 encloses asmall angle of inclination to the horizontal. The main aim of theadjustability of the sample table 2 with the eccentric 80 is to orientthe sample plane 49 relative to a focal line 101 defined by a scannerhead 50, oscillating in the X direction (in this case perpendicularly tothe plane of the drawing), of the laser scanner apparatus 1. Thisscanner head 50 moves very rapidly in the X direction and on the upperside of a separating plate 99. This separating plate has a scanningopening 90. Preferably, the scanner head 50 is sunk into this scanningopening 90, so that the light beams emanating therefrom strike thesample at a short distance, and that the scanner head 50 can receive aseffectively as possible the fluorescence emission coming from the sampleand forward it to a detector 61 or to a plurality of detectors 61,61′.

FIG. 6 is a schematic diagram with basic optical elements of the laserscanner apparatus 1 with a scanner head 50 according to a firstembodiment. The laser scanner apparatus 1 for imaging and/or measuringfluorescent samples which are located on specimen slides and treatedwith two different fluorescent dyes comprises a motor-drivable sampletable 2 with a receptacle for a sample specimen slide 10 in a sampleplane 49. A first laser 51 and a second laser 52 and also a firstoptical system 53 provide two laser beams 54,55 of differing wavelengthoriented parallel to each other and extending parallel to this plane 49.A scanner means 72 comprises a scanner head 50 which can move back andforth parallel to this plane 49 and has an optical deflection element 56for deflecting the laser beams 54,55 toward the sample. A firstobjective 57 focuses the laser beams 54,55 on the sample in the plane49. This first objective 57 has a main plane 107 arranged preferablyparallel to the sample plane 49.

A second optical system 58 guides to detectors 61,61′ the emission beambundles 59,60 which are triggered by the laser beams 54,55 on the sampleand deflected by the first objective 57 and the deflection element 56 ina direction substantially parallel to the plane 49. Two detectors 61,61′of this type detect the emission beam bundles 59,60 of differingwavelength coming from the samples. The openings of the diaphragms 48have preferably a larger diameter than the focused emission beam bundles59,60, although they can also substantially correspond to the dimensionsof the focused emission beam bundles 59,60; this would provide aconfocal laser scanner apparatus 1.

The optical deflection element 56 of the laser scanner apparatus 1according to the invention comprises a wedge-shaped dichroic mirror 62with front and rear dichroic surfaces 63,64 arranged at an intermediateangle β to each other. In this case, the wedge-shaped dichroic mirror 62is adjusted in such a way that the two laser beams 54,55 are eachreflected at one of the surfaces 63,64. In this case, the wedge-shapeddichroic mirror 62 causes through the intermediate angle β a spatialseparation of the two resulting focal points 65 and the two emissionbeam bundles 59,60 guided in the direction of the detectors 61,61′. Thetwo resulting focal points 65,65′ are arranged at a distance δ from eachother in the sample plane 49. In this first embodiment shown in FIG. 6,the optical deflection element 56 is a wedge-shaped dichroic mirror 62.Preferably, the rear dichroic surface 64 of the wedge-shaped dichroicmirror 62 is in this case configured for reflecting a first laser beam54 and the front dichroic surface 63 thereof for reflecting a secondlaser beam 55 and the two emission beam bundles 59,60.

The second optical system 58 comprises elements which are known per sesuch as a second objective 57′ which focuses the entering emission beambundles 59,60 in a respective point. The second optical system 58additionally comprises a diaphragm 48, the openings of which arepreferably substantially larger than the focused emission beam bundles59,60 passing through these openings. According to a particularlypreferred embodiment, the laser scanner apparatus 1 is thus based on anon-confocal imaging principle. According to this, these focusedemission beam bundles 59,60 each strike a detector 61,61′ which measuresthe intensity of the respective emission beam bundles 59,60. This secondobjective 57′ can be configured as an achromat or as a simple lens.

FIG. 7 shows schematic diagrams of the scanner head of the laser scannerapparatus according to the invention. In this case, FIG. 7A shows asecond embodiment of the scanner head 50 in which the optical deflectionelement 56 is configured as a pentamirror arrangement 66 with awedge-shaped dichroic mirror 62 and a simple mirror 67. As previously inthe first embodiment (cf. FIG. 6), the rear dichroic surface 64 of thewedge-shaped dichroic mirror 62 is configured for reflecting a firstlaser beam 54 and the front dichroic surface 63 thereof for reflecting asecond laser beam 55 and the two emission beam bundles 59,60.

FIG. 7B shows a third embodiment of the scanner head 50 in which theoptical deflection element 56 is likewise configured as a pentamirrorarrangement 66 with a wedge-shaped dichroic mirror 62 and a simplemirror 67. In contrast to the second embodiment (cf. FIG. 7A), thearrangement of the dichroic mirror 62 and the simple mirror 67 isinverted. In this case, the rear dichroic surface 64 of the wedge-shapeddichroic mirror 62 is configured for reflecting a first laser beam 54and the front dichroic surface 63 thereof for reflecting a second laserbeam 55 and also the first and second emission beam bundles 59,60.

In a further alternative embodiment of the scanner head 50 (not shown),the optical deflection element 56 is also configured as a pentamirrorarrangement 66 with a wedge-shaped dichroic mirror 62 and a simplemirror 67. In this case, the rear dichroic surface 64 of thewedge-shaped dichroic mirror 62 is configured for reflecting a firstlaser beam 54 and the two emission beam bundles 59,60 and the frontdichroic surface 63 thereof for reflecting a second laser beam 55.

According to a further alternative embodiment (not shown), the opticaldeflection element 56 is also configured as a pentamirror arrangement66. In contrast to the embodiment illustrated in FIG. 7A, which is perse similar, the wedge-shaped dichroic mirror 62 is replaced by a firstsimple dichroic mirror which takes the place of the front surface 63. Asecond simple dichroic mirror or a full mirror takes the place of therear surface 64 of the wedge-shaped dichroic mirror 62. The secondsimple dichroic mirror or the full mirror each encloses a correspondingintermediate angle with the first simple dichroic mirror. Thesevariations of the reflection and transmission properties of thepentamirror arrangement 66 basically also apply to embodiments withsimple mirrors arranged substantially at 45°.

It will be self-evident that the embodiments which have just beendescribed and further combinations can be swapped over as desired.Preference is nevertheless given to the first embodiment according toFIG. 6, in which the optical deflection element 56 is configured as awedge-shaped dichroic mirror 62, or to the second embodiment accordingto FIG. 7A, in which the optical deflection element 56 is configured asa pentamirror arrangement 66 with a wedge-shaped dichroic mirror 62 anda simple mirror 67. For all pentamirror arrangements 66, it is the casethat the front surface 63 and the simple mirror 67 preferably enclose anangle of 22.5°.

FIG. 8 is a horizontal partial section through a laser scanner apparatus1 with basic optical elements of a first optical system 53 for providingexcitation light and of a second optical system 58 for detecting thetriggered fluorescence emission of the samples, a scanner means 72 witha scanner head 50 and an object table 2 with a storage unit 4 comprisingspecimen slide magazines 7′,9′. Preferably, all basic optical elementsand the scanner means 72 are arranged on a common separating plate 99and the sample table 2 is arranged below this separating plate 99 (cf.FIG. 5).

The basic optical elements of the first optical system 53 are arrangedin a housing 5 and include at least a first laser 51 and optionally asecond laser 52, filter wheels 97 for the laser beams 54,55 emanatingfrom the laser or lasers 51,52 and also a number of dichroic mirrors 62and simple mirrors 67 for deflecting the laser beams 54,55 from thelasers 51,52 in a direction parallel to the X direction.

The basic optical elements of the second optical system 58 are arrangedin the same housing 5 and include one or more detectors 61,61′, filterwheels 97 and diaphragms 48 preceding said detectors for the emissionbeam bundles 59,60 emanating from the samples and also a number ofdichroic mirrors 62 and simple mirrors 67 for deflecting the emissionbeam bundles 59,60 from a direction parallel to the X direction in thedirection of the detectors 61,61′.

The scanner means 72 comprises a drive 71, the scanner head 50 andpreferably a counter oscillator 73 having a mass which is the same as orat least equivalent to the scanner head 50 for pulse compensation. Thescanner head and counter oscillator are connected to the drive 71 bymeans of connecting rods 70,70′ and each fastened to a precise linearguide (not shown). The drive 71 brings the scanner head 50 into a rapidback-and-forth movement in a direction of movement 75 (cf. the soliddouble-headed arrow) which at the same time defines the scan axis 75. Inthis case, the counter oscillator 73 performs at all times an oppositemovement, thus allowing the separating plate 99, and therefore the laserscanner apparatus 1 as a whole, to be steadied despite the preferablyhigh scanning speed of the scanner head 50. The scan axis 75 is parallelto the X axis or coincides precisely therewith. The scanner head 50comprises an optical deflection element 56 which is configured, forexample, as a dichroic mirror 62. This deflection element 56 can beconfigured as a full mirror, prism, pentaprism, pentamirrorconfiguration or as a combination of these elements listed here. Thisdeflection element 56 guides on the one hand the laser beams 54,55 ofthe first optical system 53 to the samples on the sample table 2 and onthe other hand the emission beam bundles 59,60 emitted by the samples inthe direction of the second optical system 58.

Perpendicularly to the X axis and scan axis 75, the direction ofmovement of the sample table 2 arranged below the separating plate 99extends in the direction of the Y axis. The storage unit 4 with thesample specimen slides 8 stored in a sample part magazine and the testspecimen slides 10 stored in a test part magazine 9′ is arrangedpreferably in a region outside the separating plate 99. The presence ofa specimen slide 8,10 in a specific bearing point 6 of these magazines7′,9′ is preferably checked by means of a control device 22. Thiscontrol device preferably comprises a light beam 23 which passes througha control opening 21 for these control purposes.

Preferably, the laser scanner apparatus 1 has a ventilation means 24with a fan 25, an air inlet 26 with an activated carbon filter 27 and anair outlet 28 in order to reduce the exposure to ozone of thefluorescent dyes on or in the samples stored in the sample part magazine7′. Particularly preferably, the ventilation means 24 comprises anadditional housing 29 which substantially surrounds the sample part 7with the sample specimen slides 8. This additional housing 29 ispreferably arranged within the housing 5 of the laser scanner apparatus1 and configured as a swivel-away, at least substantially closed-offregion. It is in this case particularly preferred that the ventilationmeans 24 is accommodated in this additional housing 29 and independentof the ventilation of the laser scanner apparatus 1.

A service expert can for example open this additional housing 29 and ifnecessary insert or replace one or more test specimen slides in the testpart magazine 9′ which is otherwise inaccessible. Preferably, thisadditional housing 29 is configured so that it can swivel away from theseparating plate 99 and has a charging opening 100 through which arespective specimen slide 8,10 can be trans-ported to the sample table 2or into a magazine 7′,9′. Preferably, the sample part 7 is arrangedaxially above the test part 9 of the storage unit 4 and is swiveled awaytogether with the additional housing 29 or at least together with a partof this additional housing (which is thus opened for the serviceexpert). Should a sample specimen slide 8 become jammed or break duringtransportation between the sample table 2 and the storage unit 4, theoperator can remove the defective sample specimen slide 8 withoutproviding access to the test specimen slides 10.

It is particularly preferred that a service expert slides one or moretest specimen slides 10 individually into a sample part magazine 7′ andinserts this sample part magazine 7′ into the laser scanner apparatus 1in the ordinary manner. Correspondingly programmed firmware in thecontroller 40 of the laser scanner apparatus 1 is then preferablyactivated by entering a personal identification number (PIN) of theservice expert or by entering a code for the service experts. Onceactivated in this way, the firmware enables the controller 40 of thelaser scanner apparatus 1 to control the automatic transportation ofeach of these test specimen slides 10 from the sample part magazine 7′to the sample table 2 and further into a bearing point 6 of the testpart magazine 9′. According to this particularly preferred method, anymanual intervention into the test part magazine 9′ is renderedimpossible. Only in particular emergencies and using suitable tools, aservice expert could extract the test specimen slides 10 which arepreferably enclosed in the additional housing 29. Preferably, thecontroller 40 of the laser scanner apparatus 1 according to theinvention is configured to control an automated, internal instrumentcheck which is carried out based on test specimen slides 10.

Preferably, the sample table 2 is configured so as to be motor-driven tomove up to immediately before the storage unit 4 and the position andmovement thereof are controlled by the controller 40. The same appliesalso to the adjustment plate 11 of the storage unit 4 for selecting thespecimen slide 8,10 to be examined and to the rotatable eccentric roller19 for swiveling away the flaps 16. In addition, it is preferred thatthe discharging slide 31 is also configured so as to be motor-driven fortransporting a specimen slide 8,10 to the sample table 2 for theautomated selecting and providing of a sample specimen slide 8 or testspecimen slide 10 on the sample table 2 and the position and movementthereof are controlled by the controller 40. The same applies also tothe charging slide 32 for transporting a specimen slide 8,10 to thestorage unit 4 when said storage unit is returned to a bearing point 6of the sample part magazine 7′ or the test part magazine 9′.

FIG. 9 is a horizontal partial section through the scanner head 50 ofthe laser scanner apparatus 1 with the associated displacementtransducer 91. A linear guide 68, on which the scanner head 50 isarranged so as to move in the X direction and dip into a scanningopening 90, is fastened to a frame 82. In this case, the X axiscoincides with the direction of movement 75 of the scanner head 50, thisdirection of movement 75 defining, together with the first and secondlaser beams 54,55 which are deflected toward the sample (not shown)arranged below the scanner head 50, a scanning plane 76. This scanningplane 76 stands preferably perpendicular to the sample plane 49. Thescanner head 50 comprises a measuring rod 77 which is arranged set apartfrom a fixed, linear measuring system 78 of the laser scanner apparatus1 and in this scanning plane 76. The sample table 2 is preferablyconfigured so as to be linearly movable in a Y direction, arranged atright angles to the X axis 75, of a Cartesian coordinate system andmotor-driven.

The scanner head, with all its optical elements, fastening means, themeasuring rod 77 and a part of the linear guide, has a centre of mass74. This centre of mass 74 is arranged in the direction of movement 75of the scanner head 50 on a line with a connecting rod engagement point69, which line connects the connecting rod 70 of the scanner head 50 tothe drive 71. This connecting rod engagement point 69 can be configured,for example, as an axis; it is however preferred to configure theconnecting rod engagement point as a flexural pivot.

FIG. 10 is a schematic diagram of the displacement transducer 91 for thescanner head 50 and the non-linear movement thereof during scanning asan X/t diagram. This X/t diagram points to the different periods of time(Δt₁; Δt₂) for detecting a pixel (Δx) depending on the position on the Xaxis. The displacement transducer signal 92 corresponds roughly to asine curve which has its peaks at the extreme points (end points) of ascan line of the laser head 50. Owing to the conversion of the directionof scanning in these end points and the movement which is slowed down asa result, the scanner head requires in proximity to these turning pointsa longer time (Δt₂) to cover the same distance (Δx) than at the maximumspeed of the scanner head that can be achieved in a central positionbetween the turning points, in which the same distance (Δx) is passedthrough in a much shorter time (Δt₁). The pixel (Δx) and thecorresponding location and point in time are correlated with each otherand assigned to the intensity measured at this point in time. The sum ofall measured pixels then produces a two-dimensional image. Thecorrelation of the location of these pixels in the sample plane 49 withthe intensity of the fluorescence intensity measured at this locationultimately determines, in combination with the pixel size, theresolution of the laser scanner apparatus 1.

FIG. 11 shows a test specimen slide 10 which has the format of astandard specimen slide for light-optical microscopy and which comprisesexclusively substantially light-stable test structures 41. The term“substantially light-stable” describes a test structure if saidstructure sustains no measurable damage during normal use, i.e. underradiation exposure such as conventionally occurs during testing.Irradiation for minutes or even hours of a test specimen slide 10 with alaser beam 54,55, or the leaving of a test specimen slide 10 at anunprotected location for a relatively long time (for example exposed toambient light) is not described as “normal use”.

The following Table 1 provides an overview of the most common glassspecimen slides for light-optical microscopy:

Type Imperial: 1 × 3 inches Metric: 25 × 75 mm Dimensions: Length ×breadth 76.2 mm × 25.4 mm 76 mm × 25 mm (Tolerances) (±0.5 mm) (±0.5 mm)Thickness: “standard” 1.02 mm (±0.05 mm) “thick” 1.2 mm (±0.1 mm) 1.02mm (±0.05 mm) Treatment: Corners sharp, beveled sharp, beveled Edgessharp, beveled sharp, beveled Surfaces bright, sandblasted, bright,sandblasted, on one or both sides on one or both sides

The exemplary test specimen slide 10 illustrated in FIG. 11 has a facehaving a length A of 75 mm, a breadth or width B of 25 mm and athickness C of 1 mm. One half of the face A/2 is matt-finished (forexample by means of grinding). The other half has a preferred linearpattern having a breadth or width D of 20 mm.

This linear pattern consists preferably of a vapor-deposited chromiumlayer produced by means of a mask. The upper-case letters E, F, G denotea specific number of pairs of lines per millimeter (lp/mm) and thelower-case letters l, m, n, o denote a specific mass as follows:

E=50 lp/mm; F=100 lp/mm; G=10 lp/mm;l=0.5 mm; m=2 mm; n=1 mm; o=7 mm.

All these test structures 41 are preferably exclusively substantiallylight-stable and non-fluorescent.

FIG. 12 is a vertical section through an eccentric device 80,106 foradjusting the focal line 101, which is determined by the scanner head50, relative to the sample plane 49 on the sample table 2. Eacheccentric device 80,106 is preferably motor-driven and comprises a ballbearing comprising an upright outer ring 108, a rotatable inner ring 109and a number of rolling bodies or balls 110. Preferably, a rollingbearing of this type also comprises a cage which has been omitted fromFIG. 12 for the sake of clarity. The rotatable inner ring 109 of theespecially preferred rolling bearing has an eccentric bore 111 in whicha motor-driven drive shaft 112 is fastened so as not to be able torotate relative to the inner ring 109. If then this drive shaft 112,which is fastened in a stationary and rotatable manner to a suspension(not shown), is rotated through a specific angle, the upright outer ring108, which is fastened to the moving component, is raised or lowered.This raising or lowering is determined by the eccentric mass 113 whichdefines the distance from the centre of the drive shaft 112 to thecentre of rotation 114 of the ball bearing, by the instantaneous mutualarrangement of these two centers and by the direction of rotation of thedrive shaft. The advantages of these eccentric arrangements 80,106include the fact that almost stepless and friction-free heightadjustment is facilitated. It is clear that a smaller eccentric mass 113allows lower maximum adjustability of the eccentric devices 80,106, butincreases the fineness of this adjustability. The outer ring 108 can beimmovably fastened to the device 83,103 to be moved; this is however notabsolutely necessary, so that the outer ring 108 can also be arrangedmovably relative to the device 83,103 to be moved. The eccentric mass113 can be arranged in any desired spatial direction, so that it doesnot necessarily define a horizontal deviation, such as is illustrated inFIG. 12.

The laser scanner apparatus according to the invention 1 is designed forthe imaging and measuring of two-dimensional objects. Accordingly, asensitivity calibration must also apply precisely to these “flat”objects. Two-dimensional fluorescence samples, which are bothlight-stable and chemically resistant over long periods of time, cannothowever be produced or can be produced only with difficulty.

Objects extending three-dimensionally can on the other hand be measured.However, because the intensities measured on such three-dimensionalobjects depend markedly on the depth of field of the laser scannerapparatus and on the respective positioning in the focus (i.e. in the Zdirection), three-dimensional objects of this type are not directlysuitable for calibrating signal intensity or sensitivity. There arehowever materials 102 (known as “bulk material”), such as for examplefluorescent dyes embedded in plastics material or doped glasses, whichare substantially light-stable and chemically resistant.

The spatial orientation of the sample table 2 and the storage unit ofthe laser scanner apparatus 1 are in fact arbitrary. The same applies tothe scanner means 72 which is well balanced or pulse-compensated bymeans of the counter oscillator 73. The sample plane 49 of the sampletable 2 can also be arranged substantially horizontally but hangingoverhead. Nevertheless, a stationary arrangement of the sample tableaccording to FIGS. 1 and 2 and 4 to 7 respectively is preferred. Thesame features or elements of the laser scanner apparatus 1 according tothe invention are each provided with the same reference numerals, evenif these elements are not in all cases described in detail.

Additionally disclosed is a method according to the invention foroperating a laser scanner apparatus 1 of this type for imaging and/ormeasuring fluorescent samples which are located on specimen slides andtreated with fluorescent dyes. This method is characterized in that theoptical deflection element 56 used is a wedge-shaped dichroic mirror 62with front and rear dichroic surfaces 63,64 arranged at an intermediateangle β to each other, the wedge-shaped dichroic mirror 62 beingadjusted in such a way that the two laser beams 54,55 are each reflectedat one of the surfaces 63,64, and the wedge-shaped dichroic mirror 62causing through the intermediate angle β a spatial separation both ofthe two resulting focal points 65 and of the two emission beam bundles59,60 guided in the direction of the detectors 61,61′.

This method preferably uses an optical deflection element 56 configuredas a pentamirror arrangement 66 with a wedge-shaped dichroic mirror 62and a simple mirror 67, this pentamirror arrangement 66 correctingtilting of the scanner head 50 about a Y axis extending at right anglesto the scan axis 75 in such a way that the resulting focal points 65 donot change their current position in the sample plane 49.

It is especially preferred that the scanner head 50 defines with itsoptical deflection element 56 and its direction of movement 75 ascanning plane 76 which stands perpendicular to the sample plane 49, theexcursion of the scanner head 50 in the X axis 75 being measured by ameasuring rod 77 which is arranged set apart from a linear measuringsystem 78 of the laser scanner apparatus 1 and in this scanning orscreen plane 76. This measuring rod 77 is preferably arranged in thescanning or screen plane 76 or at least in direct proximity to thisscanning plane 76. This measuring rod 77 is preferably also arranged inthe main plane 107 of the first objective 57 (cf. FIGS. 6 and 7) or atleast in direct proximity to this main plane 107.

The method according to the invention for operating a laser scannerapparatus 1 of this type for imaging and/or measuring fluorescentsamples which are located on specimen slides and treated withfluorescent dyes allows a nominal limit resolution, during the imagingand/or measuring of fluorescent samples which are located on specimenslides and treated with two different fluorescent dyes, of 2.5 μm orbetter.

Preferred is an instrument check selected from a group comprising thecarrying-out of intensity and sensitivity tests, of cross-talk,resolution and dynamic measurements, of laser noise and intensitymeasurements and of filter blocking and filter transmission checks andalso the checking of the adjustment of optical components, thegeometrical image parameters and the image orientation. Examples ofgeometrical image parameters include the magnification and theascertaining and parameterization of distortions. The superimposition oftwo or more excitation channels and/or detection channels can also bechecked. Furthermore, the laser scanner apparatus 1 is capable ofcontrolling the autofocus function.

Preferably, a sensitivity check is carried out without a fluorescentsubstance, with at least one test specimen slide 10 which has at leastapproximately the format of a standard specimen slide for light-opticalmicroscopy has and which comprises exclusively substantiallylight-stable test structures 41. Alternatively or additionally to theaforementioned sensitivity check on substantially two-dimensional teststructures 41 without a fluorescent substance, an intensity measurementcan also be carried out on three-dimensional, fluorescent teststructures:

Instead of a normal scan or a normal scanned field in the XY direction,and thus parallel to the sample plane 49, a scan is carried out in theXZ direction (Z profile) by scanning a field standing at leastsubstantially perpendicular on the sample plane 49. The directlymeasured Z profile represents the measured intensity as a function ofthe Z coordinate (I=I(Z)). Instead of this Z profile, the firstderivation of the corresponding intensities (dI=dI(z)/dz) is thencalculated, thus again providing a two-dimensional intensitydistribution. The maximum of the first derivation is thus a measure ofthe intensity measured by the laser scanner apparatus 1 at the surfaceof the sample.

The materials 102 which are suitable for this calibration process can bearranged together with the vapor-deposited linear patterns on the sametest specimen slide 10 or on a separate test specimen slide. These flat,three-dimensional materials 102 preferably have an extension parallel tothe sample plane 49 of from 2×2 mm to 10×10 mm and have a thickness offrom approximately 0.1 to 2 mm, preferably a thickness of approx. 1 mm(cf. FIG. 11).

A person skilled in the art is familiar with the function of a dichroicmirror as an optical element which is permeable to one portion of thewavelength spectrum and reflects another portion of this wavelengthspectrum. A person skilled in the art will therefore refer in this caseto wavelength-selective transmission and reflection. In addition, thereare lasers know which are capable to emit light with differentwavelengths and others (i.e. hybrid lasers) that comprise a diode lasercavity (e.g. red, 635 nm) and a solid state laser cavity (e.g. green,532 nm) in one single casing. Combinations or variants, which will beapparent to a person skilled in the art from the present description orof the described embodiments of the present invention form part of thescope thereof.

REFERENCE NUMERALS

 1 Laser scanner apparatus  2 Sample table  3 Transportation device  4Storage unit  5 Housing  6 Bearing point  7 Sample part   7′ Sample partmagazine  8 Sample specimen slide  9 Test part   9′ Test part magazine10 Test specimen slide 11 Movable adjustment plate 12 Bearing webs 13Contact spring 14 Longitudinal edge of specimen slide 15 Insertion side16 Swivel-away flap 17 Axis 18 Angular plate 19 Eccentric roller 20Locking plate 21 Control opening 22 Control device 23 Light beam 24Ventilation means 25 Fan 26 Air inlet 27 Activated carbon filter 28 Airoutlet 29 Additional housing 30 Spring 31 Discharging slide 32 Chargingslide 33 Pivotable flap 34 Receptacle 35 Opposing grooves 36 Stationarywebs 37 Movable jaw 38 Upright side walls 39 Movable contact parts 40Controller 41 Light-stable test structures 42 Handle 43 Dovetail 44Drive for 11 45 Drive for 31 46 Drive for 32 47 Tilt axis of 33 48Diaphragm 49 Plane, sample plane 50 Scanner head 51 First laser 52Second laser 53 First optical system 54 First laser beam 55 Second laserbeam 56 Optical deflection element 57 First objective  57′ Secondobjective 58 Second optical system 59 First emission beam bundle 60Second emission beam bundle 61 First detector  61′ Second detector 62Dichroic mirror 63 Front surface 64 Rear surface 65 Resulting focalpoints 66 Pentamirror arrangement 67 Simple mirror 68 Linear guide 69,69′ Connecting rod engagement point 70, 70′ Connecting rod 71 Drive 72Scanner means 73 Counter oscillator 74 Centre of mass 75 Direction ofmovement X axis, scan axis 76 Scanning plane 77 Measuring rod 78 Linearmeasuring system 79 Tilting mechanism 80 Eccentric, eccentric device 81Hinge pin 82 Frame 83 Suspension 84 Spindle drive 85 Linear guide 86Coupling 87 Motor 88 Dimpling punch 89 Ramp 90 Scanning opening 91Displacement transducer 92 Displacement transducer signal 97 Filterwheel 98 Recess 99 Separating plate 100  Charging opening 101  Focalline 102  Flat materials 103  Bearing part of 2 104  Steel spring 105 Yoke 106  Eccentric, eccentric device 107  Main plane of the objective57 108  Outer ring 109  Inner ring 110  Rolling bodies, balls 111 Eccentric bore 112  Drive shaft 113  Eccentric mass 114  Centre ofrotation of the ball bearing

1. A laser scanner apparatus (1) for imaging and/or measuringfluorescent samples which are located on specimen slides and treatedwith two different fluorescent dyes, comprising: (a) a motor-drivablesample table (2) with a receptacle (34) for specimen slides (8,10) in asample plane (49); (b) at least one laser (51,52) and a first opticalsystem (53) for providing two laser beams (54,55) of differingwavelength oriented parallel to each other and extending parallel tothis plane (49); (c) a scanner means (72) comprising a scanner head (50)which is accomplished to be moveable parallel to this plane (49) andback and forth in a direction of movement (75) and has an opticaldeflection element (56) for deflecting the laser beams (54,55) towardthe sample; (d) a first objective (57) for focusing the laser beams(54,55) on the sample in the plane (49); (e) a second optical system(58) for forwarding to detectors (61,61′) emission beam bundles (59,60)which are triggered by the laser beams (54,55) on the sample anddeflected by the first objective (57) and the deflection element (56) ina direction substantially parallel to the plane (49), and (f) twodetectors (61,61′) for detecting the emission beam bundles (59,60) ofdiffering wavelength coming from the samples, wherein the opticaldeflection element (56) comprises a wedge-shaped dichroic mirror (62)with front and rear dichroic surfaces (63,64) which are arranged at anintermediate angle (β) to each other, the wedge-shaped dichroic mirror(62) being adjusted in such a way that the two laser beams (54,55) areeach reflected at one of the surfaces (63,64), and the wedge-shapeddichroic mirror (62) causing through the intermediate angle (β) aspatial separation of the two resulting focal points (65) and the twoemission beam bundles (59,60) guided in the direction of the detectors(61,61′).
 2. The laser scanner apparatus (1) according to claim 1,wherein the optical deflection element (56) is a wedge-shaped dichroicmirror (62) or is configured as a pentamirror arrangement (66) with awedge-shaped dichroic mirror (62) and a simple mirror (67).
 3. The laserscanner apparatus (1) according to claim 1, wherein the rear dichroicsurface (64) of the wedge-shaped dichroic mirror (62) is configured forreflecting a first laser beam (54) and the front dichroic surface (63)thereof for reflecting a second laser beam (55) and the two emissionbeam bundles (59,60).
 4. The laser scanner apparatus (1) according toclaim 1, wherein the rear dichroic surface (64) of the wedge-shapeddichroic mirror (62) is configured for reflecting a first laser beam(54) and a first emission beam bundle (59) and the front dichroicsurface (63) thereof for reflecting a second laser beam (55) and asecond emission beam bundle (60).
 5. The laser scanner apparatus (1)according to claim 1, wherein the scanner head (50) is displaceablyfastened to a linear guide (68), which defines a direction of movement(75) of the scanner head (50), and has a connecting rod engagement point(69) which is connected to the drive (71) of the scanner means (72) viaa connecting rod (70).
 6. The laser scanner apparatus (1) according toclaim 5, wherein the connecting rod engagement point (69) of the scannerhead (50) is arranged in the direction of movement (75) on a line withthe centre of mass (74) of the scanner head (50).
 7. The laser scannerapparatus (1) according to claim 1, wherein the scanner head (50)defines with its optical deflection element (56) and its direction ofmovement (75) a scanning plane (76), the scanner head (50) comprising ameasuring rod (77) which is arranged set apart from a linear measuringsystem (78) of the laser scanner apparatus (1) and in this scanningplane (76).
 8. The laser scanner apparatus (1) according to claim 1,wherein the scanner head (50) comprises a measuring rod (77) which isarranged at least in proximity to the main plane (107) of the firstobjective (57).
 9. The laser scanner apparatus (1) according to claim 1,wherein the scanner means (72) comprises a counter oscillator (73) whichis displaceably fastened to a linear guide and has a connecting rodengagement point (69′) which is connected to the drive (71) of thescanner means (72) via a connecting rod (70′).
 10. The laser scannerapparatus (1) according to claim 1, wherein the sample plane (49) isarranged substantially horizontally.
 11. The laser scanner apparatus (1)according to claim 6, wherein the direction of movement (75) of thescanner head (50) defines an X axis or scan axis and the sample table(2) is linearly movable in a Y direction, arranged at right anglesthereto, of a Cartesian coordinate system.
 12. The laser scannerapparatus (1) according to claim 1, wherein the sample table (2)comprises a tilting mechanism (79) with a motor-driven eccentric (80)and a one-sided hinge pin (81), which tilting mechanism (79) allows aspecimen slide (8,10) or a sample to be oriented relative to a focalline (101).
 13. The laser scanner apparatus (1) according to claim 1,wherein the laser scanner apparatus (1) comprises a frame (82) and asuspension (83) on which the sample table (2) is moveable linearly in aY direction by means of a spindle drive (84), this suspension (83) beingpivotably fastened to the frame (82) resting on a motor-driven eccentric(80) which is carried by the frame (82) and which is accomplished to beusable for adjusting the receptacle (34) of the sample table and thusthe sample plane (49) in a substantially perpendicular Z direction. 14.The laser scanner apparatus (1) according to claim 1, wherein for thepurpose of transferring sample specimen slides (8) or test specimenslides (10), the sample table (2) is configured so as to be able to moveup to immediately before a storage unit (4) for specimen slides (8,10)of this type.
 15. The laser scanner apparatus (1) according to claim 1,wherein the receptacle (34) of the sample table (2) comprises twomutually opposing grooves (35) for receiving the two longitudinal edges(14) of a sample specimen slide (8) or a test specimen slide (10). 16.The laser scanner apparatus (1) according to claim 15, wherein thesample table (2) comprises, for securing a specimen slide (8,10) in aclamping manner in a direction substantially perpendicular to thesurface of the specimen slides, two stationary webs (36) and a jaw (37)which is accomplished to move resiliently toward these webs (36) and hastwo upright side walls (38) which define, together with the lower edgesof the webs (36), the opening width of the grooves (35).
 17. The laserscanner apparatus (1) according to claim 15, wherein the sample table(2) comprises, for securing a specimen slide (8,10) in a clamping mannerin a direction substantially parallel to the surface of the specimenslides, contact parts (39) which are moveable toward at least one of thelongitudinal edges (14) of the specimen slide and resiliently delimitthe opening breadth of the receptacle (34).
 18. The laser scannerapparatus (1) according to claim 17, wherein the contact parts (39),which are moveable toward at least one of the longitudinal edges (14) ofthe specimen slide, are configured as rolls each having a substantiallyvertical axis.
 19. The laser scanner apparatus (1) according to claim14, wherein the storage unit (4) comprises a respective sample part (7),which has at least one respective bearing point (6) and is accessibleduring operation of the laser scanner apparatus (1) to a transportationdevice (3), for sample specimen slides (8) and test part (9) for testspecimen slides (10), the test part (9) being configured separately fromthe sample part (7) and as a test part magazine (9′), which is rigidlyconnected to the laser scanner apparatus (1), for one or more testspecimen slides (10), as a result of which a test specimen slide (10)stored in the test part (9) is not manually accessible to an operatorwhen the laser scanner apparatus (1) is in operation.
 20. The laserscanner apparatus (1) according to claim 1, wherein the laser scannerapparatus (1) comprises a controller (40) which is configured forcontrolling an automated, internal instrument check carried out based ontest specimen slides (10).
 21. A method for operating a laser scannerapparatus (1) for imaging and/or measuring fluorescent samples which arelocated on specimen slides and treated with two different fluorescentdyes, which method includes the following steps: (a) providing amotor-drivable sample table (2) with a receptacle (34) for specimenslides (8,10) in a sample plane (49); (b) providing two laser beams(54,55) of differing wavelength oriented parallel to each other andextending parallel to this plane (49) with at least one laser (51,52)and a first optical system (53); (c) deflecting the laser beams (54,55)toward the sample with an optical deflection element (56) of a scannermeans (72) comprising a scanner head (50) which is moveable back andforth parallel to this plane (49) and in a direction of movement (75);(d) focusing the laser beams (54,55) on the sample in the plane (49)with a first objective (57); (e) forwarding to detectors (61,61′)emission beam bundles (59,60) which are triggered by the laser beams(54,55) on the sample and deflected by the first objective (57) and thedeflection element (56) in a direction substantially parallel to theplane (49) with a second optical system (58), and (f) detecting theemission beam bundles (59,60) of differing wavelength coming from thesamples using two detectors (61,61′), wherein the optical deflectionelement (56) used is a wedge-shaped dichroic mirror (62) with front andrear dichroic surfaces (63,64) arranged at an intermediate angle (β) toeach other, the wedge-shaped dichroic mirror (62) being adjusted in sucha way that the two laser beams (54,55) are each reflected at one of thesurfaces (63,64), and the wedge-shaped dichroic mirror (62) causingthrough the intermediate angle (β) a spatial separation of the tworesulting focal points (65) and the two emission beam bundles (59,60)guided in the direction of the detectors (61,61′).
 22. The methodaccording to claim 21, wherein an optical deflection element (56)configured as a pentamirror arrangement (66) with a wedge-shapeddichroic mirror (62) and a simple mirror (67) is used, this pentamirrorarrangement (66) correcting tilting of the scanner head (50) about an Yaxis extending at right angles to the scan axis (75) in such a way thatthe resulting focal points (65) do not change their current position inthe sample plane (49).
 23. The method according to claim 21, wherein thescanner head (50) defines with its optical deflection element (56) andits direction of movement (75) a scanning plane (76), the excursion ofthe scanner head (50) in the X axis (75) being measured with a measuringrod (77) which is arranged set apart from a linear measuring system (78)of the laser scanner apparatus (1) in this scanning plane (76).
 24. Themethod according to claim 21, wherein the excursion of the scanner head(50) in the X axis (75) is measured with a measuring rod (77) which isarranged at least in proximity to the main plane (107) of the firstobjective (57).
 25. The method according to claim 21, wherein thenominal limit resolution during the imaging and/or measuring offluorescent samples which are located on specimen slides and treatedwith two different fluorescent dyes is 2.5 μm or better.